Wnt5 protein and mRNA expression domains in epidermis, muscle and tendon cells during embryonic development.

<p>WNT5 is predominantly expressed in subsets of neurons in the CNS from stage 12 onwards throughout embryonic development (data not shown; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone.0032297-Fradkin2" target="_blank">[28]</a>). However, there is also strong expression from this stage onwards in the epidermis and the musculature. At stage 12, Wnt5 protein (<b>A, B</b>) and <i>Wnt5</i> mRNA (<b>E, F</b>) expression is observed in the epidermis, most prominently in two clusters (<b>arrows</b>), and throughout the somatic mesoderm that will give rise to the body wall musculature. Later in embryonic development at early stage 16 WNT5 protein and <i>Wnt5</i> mRNA are present in the attachment sites (<b>arrows in panels C and G</b>) and at low levels in most muscle fibers including the LTMs 21, 22 and 23 (<b>C, G</b>). At the end of embryonic development at late stage 17, Wnt5 protein (<b>D</b>) and <i>Wnt5</i> mRNA (<b>H</b>) are almost undetectable in the somatic mesoderm. In all panels anterior is up and ventral is left.</p

The new attachment sites of the bypassed muscle fibers in <i>Wnt5</i> and <i>drl</i> mutants frequently do not express SR, while the bypassed attachment sites do.

<p>Double labeled stage 16 embryos are shown of <i>w¹¹¹⁸</i> (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^Red2</i> (<b>C</b>) with anti-Muscle Myosin in green and anti-SR in red (Material and Methods). Asterisks mark the novel attachment sites of the overshooting LTM muscles; white arrowheads mark the locations of the original attachment sites. In <i>Wnt5</i> mutants the novel target sites do not express SR in 65% of the segments containing overshooting muscles, while the bypassed attachment sites usually express SR. The SR positive, original tendon cell is also present in <i>drl^Red2</i> mutants, but is partly masked by the overshooting muscle fiber in panel (<b>C</b>), but clearly visible in panel (F)). These results were confirmed in embryos that express Tau-MYC under the control of a <i>stripe</i> promoter in both <i>Wnt5</i> and <i>drl</i> mutants (data not shown). The following genotypes are shown, the control UAS-Tau-MYC; <i>sr</i>-GAL4 embryos (<b>D</b>), <i>Wnt5⁴⁰⁰</i>; UAS-Tau-MYC/<i>sr</i>-GAL4 (<b>E</b>) and <i>drl^Red2</i>; UAS-Tau-MYC/<i>sr</i>-Gal4 (<b>F</b>). Anti-Muscle Myosin is shown in green and anti-MYC in red. No MYC protein is observed in the ectopic attachment sites. The photographs in Panels (<b>A</b>–<b>C</b>) were taken on a compound microscope and those in Panels (<b>D–F</b>) on a confocal microscope. Anterior is up and ventral is left.</p

LTM muscle fibers 21, 22 and 23 frequently overshoot their attachment sites in <i>Wnt5</i>, <i>drl</i> and <i>dnt</i> mutant embryos.

<p>Stage 16 embryo body wall muscle preparations stained with anti-Muscle Myosin are shown for the wild type control (<i>w¹¹¹⁸</i>) (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>), <i>drl^Red2</i> (<b>C</b>), <i>Drl-2^E124</i> (<b>D</b>), <i>dnt^42.3</i> (<b>E</b>) and Df(2L)Exel6043 (<b>F</b>). Two hemisegments are displayed for each genotype with one set of muscles 21–23 labelled. In <i>Wnt5</i>, <i>drl</i> and <i>dnt</i> mutants, LTMs frequently bypass their normal attachment at the epidermis at muscle 12 and instead extend ventrally beyond muscle 13 and attach at a novel epidermal site located close to muscle fiber 7. Df(2L)Exel6043 mutant embryos, that lack both DNT and DRL, display this phenotype in all hemisegments of the homozygous animals. The penetrance of these phenotypes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone-0032297-t001" target="_blank"><b>Table 1</b></a>. The muscle bypass phenotype is schematically shown in panel (<b>G</b>). The * indicates the location of the novel, ectopic epidermal attachment in panels (<b>B</b>), (<b>C</b>), (<b>E</b>), (<b>F</b>) and (<b>G</b>). Anterior is up and ventral is left.</p

The new attachment sites of the bypassed muscle fibers in <i>Wnt5</i> and <i>drl</i> mutants express βPS integrin.

<p>Wild type (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^red2</i> (<b>C</b>) embryos were labelled with anti-βPS Integrin. Muscles 21–23 do exhibit an accumulation of βPS Integrin protein at the tip of the overshooting fibers (white asterix).</p

Muscle attachment defects persist from the embryonic to larval stages in <i>Wnt5</i> and <i>drl</i> mutants.

<p>Third instar larval body walls of <i>w¹¹¹⁸</i> (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^Red2</i> (<b>C</b>) mutant larvae are stained with anti-FAS2 (mAb 1D4). <i>Wnt5⁴⁰⁰</i> larvae and <i>drl^Red2</i> larvae frequently bypass their normal attachment sites and extend ventrally where they form new stable attachments. The original and ectopic tendons cells are indicated by + and *, respectively. FAS2 protein is evident at both sites. The penetrance of the bypass phenotypes is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone-0032297-t001" target="_blank"><b>Table 1</b></a>. Anterior is up and ventral is left.</p

Impaired Vps13 function leads to defects in protein homeostasis.

<p>(A) Percentage of isogenic control and <i>Vps13</i> mutant flies that eclosed at increasing temperatures. (B) Percentage of homozygous <i>Vps13</i> mutant flies and excision line flies that eclosed at 29°C. (C) Percentage of flies of various genotypes that eclosed at 29°C. Two independent deficiency lines (lacking a genomic area containing the <i>Vps13</i> gene) were crossed with <i>Vps13/ CyO</i> heterozygous flies. Eclosion rate of the following genotypes was analyzed: <i>Vps13/+</i>, <i>Df #7535/+</i>, <i>Vps13/Df #7535</i>, <i>Df #7534/+</i> and <i>Vps13/Df #7534</i>. (D) Percentage of <i>Vps13</i> flies that eclosed at 22°C on food with increasing concentrations of L-canavanine. (E) Western blot analysis of lysates of 1 day old control and <i>Vps13</i> mutant fly heads. Ubiquitylated proteins, K48 ubiquitylated proteins and K63 ubiquitylated proteins were detected. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p

<i>Vps13</i> mutant flies show a decreased life span, age dependent impairment of locomotor function and neurodegeneration.

<p>(A) Life span analysis of isogenic control and <i>Vps13</i> mutant flies. (B) The fraction of dead flies of total flies used,observed within the indicated time intervals. (C) Life span curve of <i>Vps13</i> mutant flies and three excision lines. (D) Climbing behavior was analyzed by determining the percentage of isogenic control and <i>Vps13</i> mutant flies (4 and 17 days old) able to climb 5 cm against gravity within 15 seconds. Mean and SEM are plotted (n = 5). For statistical analysis a two-tailed students T-test was used. P<0.001 is ***. (E) Fly heads (20 day old) of control and homozygous <i>Vps13</i> mutant flies were fixed, dehydrated and embedded in epon. Sections, visualizing a cross section of the complete brain, were stained with toluidine blue. The scale bar indicates 50 μm.(F) Higher magnification images of the boxed area’s in Fig E. The central complex is denoted with a dotted line. The scale bar indicates 25 μm.</p

Central nervous system of larval and adult <i>Vps13</i> mutants contain protein aggregates.

<p>(A) Ventral nerve cords of control, <i>Vps13</i> mutant, <i>Vps13/Df #7534</i> and <i>Vps13/Df #7535</i> third instar larvae were stained for ubiquitylated proteins and DAPI. The presence of DAPI indicates areas where nuclei of neuronal cell bodies or glial cells are located. DAPI negative regions represent areas mainly containing axonal and synaptic structures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170106#pone.0170106.ref033" target="_blank">33</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170106#pone.0170106.ref035" target="_blank">35</a>]. The areas in the grey boxes are shown below as higher magnification images. The scale bar indicates 50 μm. (B) Quantification of the number of ubiquitylated protein puncta in the ventral nerve cord. (C) Staining of 1 day old adult control brains using DAPI. The grey box denotes the area in the brain where the two antennal lobes are located. The presence of DAPI indicates areas where nuclei of neuronal or glial cell bodies are located. The center area which is negative for DAPI contains axons and synaptic structures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170106#pone.0170106.ref034" target="_blank">34</a>]. The scale bar indicates 50 μm. (D) Quantification of the number of puncta of ubiquitylated proteins in the antennal lobes derived from 1 day old isogenic controls, <i>Vps13</i> mutants and excision line 3. (E) Staining of brains derived from 1 day old controls, <i>Vps13</i> mutants and excision line 3 flies for ubiquitylated proteins, Ref(2)p and DAPI. The scale bar indicates 20 μm Arrows indicate colocalization of Ref(2)P and Ubiquitin positive foci. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p

<i>Vps13</i>^{<i>c03628</i>} encodes for a truncated Vps13 protein.

<p>(A) Schematic representation of the <i>Vps13</i> gene and the genomic localization, RNA and protein is depicted. The epitopes of the polyclonal Vps13 antibodies (Vps13 NT and Vps13 #62) are indicated.(B) Relative levels of <i>Vps13</i> mRNA in control and <i>Vps13</i> mutant flies were determined by Q-PCR. Mean and SEM (n = 2) are plotted. (C) Western blot analysis of Vps13 protein in control and <i>Vps13</i> mutant fly heads using the Vps13 #62 antibody. β-Actin was used as a loading control. (D) Western blot analysis of the level of Vps13 protein in control and <i>Vps13</i> mutant fly head extracts analyzed with the Vps13 NT antibody. α-tubulin was used as a loading control. (E) Lysates of the heads of control flies and whole control flies were analyzed for Vps13 levels. α-tubulin was used as a loading control. (F) Lysates of the heads of control flies, <i>Vps13</i> mutant flies and three excision lines were analyzed for Vps13 levels. Human VPS13A was detected in samples of Hek293 cells. <i>Drosophila</i> samples and human samples were run on the same gel, separated by a lane containing the molecular weight standards, after transfer of the membrane, the marker lane was split to detect human and Drosophila VPS13 separately using species specific antibodies. The marker lane was used to align the blots after antibody detection. α-tubulin was used as a loading control.</p

Overexpression of HsVps13A rescues phenotypes of <i>Vps13</i> mutants.

<p>(A) Samples from fly heads of <i>Actin-GAL4 / +</i> (as a control) and <i>Actin-GAL4 / UAS-HsVps13A</i> (HsVps13A expressing) flies were separated into a membrane and cytosol fraction and analyzed by Western blot for HsVps13A levels. EGFR and GAPDH used as controls for membrane and cytosolic proteins, respectively. (B) Eclosion rate of <i>Vps13</i> mutant flies a <i>Actin-GAL4/+</i> (control) or <i>Actin-GAL4/UAS-HsVp13A (HsVps13A expressing)</i> background at 25°C. (C) Ubiquitylated proteins from samples of 1 day old fly head extracts <i>of Vps13/CyO; Actin-GAL4/+</i> (as a control), <i>Vps13/ Vps13; Actin-GAL4/+</i> (representing homozygous mutants) and <i>Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A</i> (representing homozygous mutants expressing human VPS13A). (D) Representative picture of ubiquitylated protein staining of the third instar larval ventral nerve cord of <i>Vps13/CyO; Actin-GAL4/+</i> (as a control), <i>Vps13/ Vps13; Actin-GAL4/+</i> and <i>Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A</i>. Arrows indicate accumulations of ubiquitylated positive structures. The scale bar indicates 50 μm and 12,5 μm in the enlargement. (E) Quantification of the number of puncta in third instar larval ventral nerve cord of the experiment presented in Fig 6D. (F) Life span curve of <i>Vps13/ Vps13; Actin-GAL4/+</i> and <i>Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A</i>. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p